The following is a procedure for the in vitro modification of DNA before electrotransformation into ''Lactobacillus plantarum'' developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including ''Lactobacillus plantarum'' is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium.

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The following is a procedure for the in vitro modification of DNA before electrotransformation into ''Lactobacillus plantarum'' developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including ''Lactobacillus plantarum'' is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium. See notes for an alternative method.

==Materials==

==Materials==

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*10 mg ml BSA

*10 mg ml BSA

*Filtered glycerol

*Filtered glycerol

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*Prepared Plasmid DNA

*Wash Buffer (15mL)

*Wash Buffer (15mL)

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**15mg NaCl (50mM)

**15mg NaCl (50mM)

**15mg EDTA (10mM)

**15mg EDTA (10mM)

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*Overnight culture of L. plantarum, OD=1.5-2.0

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*Prepared Plasmid DNA

==Procedure==

==Procedure==

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===DNA Modification===

===DNA Modification===

1. Add the following to a 25μL aliquot of cell extract:

1. Add the following to a 25μL aliquot of cell extract:

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** 50μL TNE Solution

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** 50μL TNE Buffer

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** 10μL of S-adenosylmethionine

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** 10μL of S-adenosylmethionine Stock Solution

** 1μL BSA (10mg/ml)

** 1μL BSA (10mg/ml)

** 10μL of plasmid DNA.

** 10μL of plasmid DNA.

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==Notes==

==Notes==

All questions, input and feedback are welcome!

All questions, input and feedback are welcome!

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#AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.

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*AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.

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#AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.

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*AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.

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#There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].

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*There is a helpful protocol for phenol extraction posted[http://openwetware.org/wiki/Phenol/chloroform_extraction] and a protocol for ethanol precipitation posted[http://openwetware.org/wiki/Nucleic_acid_precipitation].

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*An alternative to this protocol is to use a lab strain of ''Lactococcus lactis'' (we use strain MG1363) as a shuttle species. The procedure takes just as much ''linear'' time, but much less actual time; and is much easier. The process goes as follows.

Current revision

Contents

Overview

The following is a procedure for the in vitro modification of DNA before electrotransformation into Lactobacillus plantarum developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including Lactobacillus plantarum is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium. See notes for an alternative method.

DNA Modification

2. Incubate the mixture at 30°C for 16 hours.
3. Extract the mixture with a phenol/chloroform extraction.
4. Precipitate using ethanol.

Notes

All questions, input and feedback are welcome!

AEBSF should be handled in a fume hood with lab coat, safety gloves and eye protection.

AEBSF is a much safer alternative to PMSF that is soluble in water and has a very similar specificity to PMSF as a serine protease inhibitor. It also goes by the name Pefabloc SC.

There is a helpful protocol for phenol extraction posted[1] and a protocol for ethanol precipitation posted[2].

An alternative to this protocol is to use a lab strain of Lactococcus lactis (we use strain MG1363) as a shuttle species. The procedure takes just as much linear time, but much less actual time; and is much easier. The process goes as follows.

Miniprep the desired shuttle vector from E. coli.

Electroporate into L. lactis electro-comptent cells at 10,000kv/cm.

Let cells recover in 25ml GM17 media for one hour.

Add the appropriate antibiotic to the media.

Grow overnight at 30°C.

Miniprep L. lactis culture.

Transform L. plantarum electro-competent cells at 10,000kv/cm.

Smile because you didn't have to buy any extra reagents or work with the loud-ass sonicator!